MOLECULAR GENETICS OF C OXIDASE

C氧化酶的分子遗传学

• 批准号: 2185985
• 负责人: LAWRENCE GROSSMAN
• 金额: $ 16.83万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-02-01 至 1997-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185985
• 关键词: cell type; cytochrome oxidase; enzyme mechanism; gene deletion mutation; gene expression; genetic regulation; genetic regulatory element; genetic transcription; immunocytochemistry; in situ hybridization; mitochondria; molecular genetics; muscle proteins; myoblasts; myotubes; oxygen tension; protein isoforms; reporter genes; site directed mutagenesis; thyroid hormones; transcription factor; transfection

Cytochrome c oxidase, the terminal protein of the electron transport chain, is composed in mammals of thirteen subunits, three encoded by mitochondrial DNA and the remainder by nuclear DNA. At least three of these nuclear subunits occur in more than one molecular form, isoforms, the products of separate genes. The proposed experiments will provide insights into the cellular function of these isoforms, which may be related to both ontogenetic development and cellular physiology. In addition, isoform expression has been implicated in human neuromuscular disease. This project will focus on two subunits, COX VIIa and COX VIIc, encoded by three genes. COX7a displays a heart- and muscle-specific isoform gene, COX7a-H, which is already isolated, and an isoform gene expressed in all tissues, COX7a-L. A third gene, COX7c, appears to have a single expressed form in all tissues. The specific aims are to (1) isolate and characterize the COX7a-L and COX7c genes, and compare the regulatory regions to those of COX7a-H; (2) make reporter constructs of each gene to define cis-acting regulatory elements by deletional and mutational approaches; (3) investigate the effect of physiological conditions on regulation of isoform expression; and (4) isolate and study trans-acting regulatory factors. The third aim, studying regulation of isoform expression, will be pursued in two ways: by in situ hybridization of isoform-specific probes to regions of the heart that operate under very different oxidative stresses, and by cotransfection of both isoform genes in distinguishable reporter constructs into cells placed under a variety of physiological conditions. This project fits into the longer term objectives of deducing the role of the nuclear- encoded subunits and the mechanisms used to regulate oxidative function, and applying this knowledge to the study of nuclear genes in mitochondrial disease.

细胞色素c氧化酶，电子传递的末端蛋白 链，在哺乳动物中由13个亚基组成，其中3个由 线粒体DNA和核DNA的其余部分。 中的至少三 这些核亚单位以一种以上的分子形式，同种型， 不同基因的产物 这些实验将提供 深入了解这些亚型的细胞功能，这可能是 与个体发育和细胞生理学有关。 在 此外，同种型表达与人类神经肌肉细胞中的 疾病 该项目将侧重于两个分单位，考克斯VIIa和考克斯VIIc， 由三个基因编码 COX 7a显示心脏和肌肉特异性 已经分离的同种型基因COX 7a-H和同种型基因 在所有组织中表达，COX 7a-L。 第三个基因，COX 7 c，似乎有 在所有组织中的单一表达形式。 具体目标是：（1） 分离并鉴定COX 7a-L和COX 7 c基因，并比较COX 7a-L和COX 7 c基因的表达。 （2）制备C 0X 7a-H的报告基因构建体， 每个基因通过缺失和缺失来定义顺式作用调节元件， （3）研究生理突变的影响 异构体表达调控的条件;（4）分离和研究 反式调节因子。 第三个目的，研究 同种型表达，将以两种方式进行：通过原位 同种型特异性探针与心脏区域的杂交， 在非常不同的氧化应激下运作， 将两种同种型基因以可区分的报告基因构建体导入细胞 置于各种生理条件下。 这个项目很适合 从长远来看，核武器的作用... 编码的亚基和用于调节氧化功能的机制， 并将这一知识应用于核基因的研究， 线粒体疾病